To transfer safely and efficiently therapeutic DNA into the central nervous system, is a formidable challenge in the development of active therapies in brain diseases.
Preliminary investigations have been carried out with a number of vectors, more particularly retroviral vectors and herpes simplex derived vectors. However the usefulness of such gene transfer vehicles has, to date, been limited.
In most cases, retroviral vectors are not useful because they are unable to infect postmitotic cells, including most neural cells (1). Herpes simplex derived vectors infect neural cells but problems of pathogenicity and of stability of gene expression remain unsolved (2,3). In addition Herpes simplex derived vectors have so far proven to have but limited efficacy of expression. In a most recent article (4) the authors refer to the short-term expression reported earlier of HSV-1 derived vectors, because most of the promoters used had only been active during the acute phase of viral infection (less than 10 days post-infection). They disclosed the expression of a b-glucuronidase gene in a cell of the central nervous system under the control of the LAT promoter normally associated with the latency-associated-transcript (LAT) sequence of the virus. But the authors also report that, even though their experiments demonstrated the feasibility of using the LAT promoter for long-term expression of foreign genes in cells of the central nervous system to correct a genetic enzymatic deficiency in infected cells, too few cells had been corrected to alter the disease phenotype. Consequently their vector system needed to be improved to correct sufficient cells for obtaining a clinically significant effect.
The invention aims at obviating such difficulties and at providing most effective vector systems capable of delivering foreign genes and, where appropriate, their transcription products or expression products directly to cells of the central nervous system, particularly to terminally differentiated cells, incapable of proliferation. A more particular object of the invention is also to allow for the wide spreading of such vector systems throughout the neural tissue to be infected, yet while remaining substantially confined thereto.
Still another object of the invention is to produce such vector systems which are sufficiently safe to allow for a study and regulation in vitro of cloned genes in such cells or in test animals, and for therapy, in man or animal, involving the in situ production of a selected expression product, including gene therapy.
The invention is based on the recognition that adenovirus-derived vectors, particularly non-replicative adenovirus vectors, are capable of fulfilling these aims, both in vitro and in vivo. They provide powerful delivery systems of genes into the cells of the central nervous system, more particularly brain cells. They are characterized by a degree of infectivity of sufficient magnitude to allow for the infection of considerable populations of cells The biological experiments disclosed hereafter demonstrate the capability of adenovirus derived vectors (or adenoviral vectors) of efficiently infecting nerve cells, particularly neurons, both in vitro and in vivo.
Thus the invention provides a process for the production of a recombinant vector useful in a method comprising causing the transcription product or the expression product of a nucleotide sequence coding for a selected polypeptide to be targeted or produced in cells of the central nervous system, e.g. brain cells, particularly neural, glial or ependymal cells, wherein said recombinant vector is an adenoviral vector which comprises at least part of the genome of an adenovirus including those regions of that genome which provide the genetic information required by that adenovirus to penetrate into cells normally infectable by it, said nucleotide sequence being inserted in said genome part, under the control of a promoter either present or also inserted within said adenoviral vector, and said promoter being operative in said cells.
Thus the invention is more particularly related to the use of an adenovirus-derived vector for the expression of a selected nucleotide in the cells of the central nervous system.
The invention also provides a recombinant DNA vector characterized in that it is capable of directing the expression and/or transcription of a selected nucleotide sequence in the cells of the central nervous system and in that it comprises (i) at least part of the genome of an adenovirus, including the regions required for that adenovirus to penetrate into the cells of the central nervous system, and (ii) said selected nucleotide sequence under the control of a promoter operative in said cells.
The powerful capability of adenoviral vectors of transferring gene fragments in vivo into quiescent neural cells is illustrated by the experiments reported hereafter, which were carried out with an adenovirus vector carrying the E. Coli lac Z gene or the human tyrosine hydroxylase gene, in neural cells of adult rats. A large number of neural cells (including neurons, astrocytes, microglia and ependymal cells) expressed these transgenes at least 60 days after inoculation of various brain areas. Injecting up to 3xc3x97105 pfu in 10 xcexcl did not result in any detectable cytopathic effects, which were only observed for the highest titers of infection ( greater than 107 pfu/10 xcexcl) and were most likely associated with a massive endocytosis of viral particles in neural cells close to the injection site.
Moreover the genomes of adenoviruses can be manipulated to accommodate foreign genes of 7.5 kb in length or more. It has a large host range, a low pathogenicity in man, and high titers of the virus can be obtained (5).
It will be readily appreciated that these results strongly support the presumption that adenovirus offers, as a vector, a new and remarkable tool to genetically modify quiescent cells of the nervous system, because of its great efficacy of infection, long term expression, wide host range and low toxicity. Thus, adenovirus should be instrumental in the study of the function of cloned gene products in their physiological and anatomical context. The ability to infect the hippocampus (as this will be shown later) is of great interest to study integrated phenomena such as long-term potentiation, in animals.
Moreover the adenovirus clearly appears as an efficient means to transfer foreign genes into the brain with a therapeutic goal. Adenovirus vectors have great potential for gene therapy of nervous system diseases, such as the local delivery of growth factors or neurotransmitters for degenerative diseases and, more generally, to replace defective genes in appropriate cells. Relatively low titers of adenovirus vectors can efficiently transfer foreign genes into a significant number of brain cells without triggerring pathological effects. Subject to optimization of the doses of recombinant adenoviral vectors containing a foreign gene to be delivered into brain cells, they open new avenues in the treatment of many genetic and acquired neurological diseases, consequently, an alternative to drug treatment or brain transplantation of fetal tissues.
Adenovires, particularly adenoviruses of type 2 or 5 (Ad2 or Ad5) are particularly preferred. They are relatively stable, can be cultured easily and rapidly (viral cycle of about 30 hours) and provide high titers: up to 104-105 plaque forming units (p.f.u.) per infected cell. They are not oncogenic. The complete sequence of their viral genome has been established (6) and its molecular biology has been studied extensively. Finally several mutants, particularly deletion mutants, have been obtained, which makes it possible to insert fragments of large size therein (7).
Preferably the recombinant vectors for use in this invention are defective adenoviruses, whose genomes no longer contain those nucleotides sequences required for the virus replication in cells (other than brain cells) normally infectable by it. More particularly, they are free of the E1 region, including the early E1a region which activates the other early transcription units of the virus required for its replication as well as the region E1b involved in the establishment of a fully transformed phenotype, in the protection of DNA sequences during viral infection and required for the normal progression of viral events late in infection.
Preferably too, they are devoid of the E3 region which is involved in cellular immunity in vivo and is totally dispensible for growth in vitro.
Preferably however the recombinant vector for use in that invention comprises all of the sequences that are essential for the adenovirus encapsidation.
The promoter controlling the sequence coding for the selected polypeptide to be targeted or produced in the cells of the central nervous system, can either be endogenous or exogenous with respect to the adenoviral parts of the recombinant vector.
A preferred homologous promoter is any one that is likely to be recognized by the polymerases, practically RNA polymerase II, of human or animal cells infected by such adenoviruses. A particularly preferred endogenous promoter is the major late strong promoter (MLP) of the human adenovirus of type 2 (Levrero et al., 1991) (8). Another promoter that may be used consists of the early promoter of the E1a region of the adenovirus. In that last instance, a preferred defective adenovirus is devoid of its 5xe2x80x2 region normally upstream for that early promoter. In that last instance, the nucleotide sequence sought to be introduced in the neural cells is substituted for the E1A region.
The endogenous promoters of the adenoviruses can also be replaced by other ubiquitary promoters of heterologous or exogenous origin, e.g.:
a promoter contained in the LTR (Long Terminal Repeat) of the Rous Sarcome Virus (RSV) or the LAT promoter referred to above,
a promoter of the IE gene of Cytomegalovirus (CMV),
inducible MMTV promoters (originating from the mouse mammary tumor virus) or metallothionine promoters.
Other promoters can be used too. Particularly, neural or glial promoters will be preferred, particularly in instances where the nucleotide sequence inserted in the adenoviral vector is to be targeted more specifically on more specific classes of neural cells. Reference is for instance made to the following promoters, particularly those involved in the genes coding for neurotransmitter synthesizing enzymes:
TH (tyrosine hydroxylase)
CHAT (choline acetyl transferase)
TPH (tryptophane hydroxylase)
GFAP (glial fibrillary acidic protein) enolase g (neuronal protein marker) aldolase C.
The invention relates also to a process for making such recombinant vectors, which process comprises inserting the nucleotide sequence whose expression is sought in the starting vectors and the transformation of infectable cells with said vectors. Where the vector is a full live virus, the recombinant viruses are then recovered from the cell culture medium. Where the recombinant vector is a defective virus, a preferred process then comprises the transformation of a transformable eucaryotic cell line (preferably of human or animal origin) which itself comprises a distinct nucleotide sequence capable of complementing the part of the adenovirus genome that is essential for its replication and which is not present in said vector, whereby that complementation sequence is preferably incorporated into the genome of said cell line.
By way of preferred examples of such cell lines, one should mention the so called xe2x80x9c293 cell linexe2x80x9d derived from human embryonary kidney which contains, integrated in its genome, the first 11% of the 5xe2x80x2 region of an Ad5 virus genome. That portion of the Ad5 genome enables recombinant viruses defective in that region, because of a deletion of part that region, to be appropriately complemented. Such a process for the production of defective viruses has been described more particularly in European patent application nxc2x0 EP 185573, filed on Nov. 20, 1985.
After transformation of such cell lines, the defective recombinant viruses are multiplied, recovered and purified.
Needless to say that the same process may be applicable to the production of other defective adenoviruses as a result of a deletion in a region other than in the 5xe2x80x2 region referred to hereabove, it being then understood that the cell lines used in such production should then include in their own genome the sequence deleted form the adenoviral genome to thereby allow for the complementation of such defective adenoviruses.
The invention provides thus for the first time a serious alternative to injection in the brain area of cells, e.g. embryonic cells carrying the relevant genetic information, in gene therapy aiming at correcting metabolic deficiencies or defects in the targeted cells, particularly post-mitotic neurons. This is the consequence of the important infective power of adenoviruses, also retained by the corresponding defective adenovirus-derived vectors, of their capacity of spreading throughout the targeted neural or nerve tissue while also remaining substantially confined within the selected tissue, if specifically injected thereinto, as well as of the long-term transcription, and in most instances, expression of the nucleotide sequence carried into the nerve tissue by such adenoviral vectors.
The nucleotide sequence whose introduction in the cells of the central nervous system may be sought, may consist of any sequence capable of providing molecules interacting with the metabolism of such cells. Such molecules may consist of selected anti-sense RNAs, or anti-sense oligoribonucleotide, capable of interacting with defective messenger RNAs whose further processing, responsible for corresponding diseases, ought to be blocked, e.g. in a number of neuro-psychiatric diseases, epilepsy, etc . . . This methodology appears of particular interest in the treatment of the Alzheimer""s disease. The anti-sense method could be used, by way of example only, for inducing a blockade of the b-amyloid precursor, for preventing an accumulation of the b-amyloid peptide in the senile plaques. Alternatively use could be made of anti-sense oligonucleotide capable of inhibiting the expression of enzymes involved in the abnormal phosphorylation of proteins, e.g. the TAU protein involved in Alzheimer""s disease. Alternatively again the nucleotide sequence is one which could sequester specific binding proteins, themselves normally involved in the processing of the DNA or RNA sequence whose transcription or expression is sought to be inhibited.
The nucleotide sequence whose introduction in the cells of the central nervous system may be sought, may also code for an expression product having a biological property. Said expression product may for example be capable (1) of compensating a corresponding defective natural polypeptide containing product encoded by a defective corresponding nucleotide sequence, or (2) of compensating the lack of endogenous production of the natural endogenous corresponding polypeptide containing product in said targeted cells, or (3) of introducing new therapeutic activities in the infected cells. Examples of such defective polypeptide-containing products may consist of neurotransmitter-synthesizing enzymes and growth factors. For example in the case of Parkinson""s disease (which is characterized by a vulnerability of dopaminergic cells) one could envisage producing locally DOPA or dopamine by expressing the cDNA encoding tyrosine hydroxylase, or a growth factor such as BDNF (brain derived neurotrophic factor) which could favor the survival of dopaminergic neurons. Likewise for Alzheimer""s disease, where one of the missing neurotransmitters is acetylcholine which is synthesized by choline acetyl transferase. Moreover NGF (nerve growth factor) could prevent degeneration of cholinergic neurons.
Another potentially useful factor to express is CNTF (ciliary neurotrophic factor) which could prevent neuron death. But CNTF may also have interesting effect in the brain, e.g. for the blocking of the destruction (seemingly induced in diabetes affected patients) of peripheral nerves. Other trophic factor whose expression may be sought consist, by way of examples of IGF, GMF, aFGF, bFGF, NT3 and NT5.
In a general manner growth factors could be caused to be produced in neuronal cells of patients affected with neuropathies, strokes, spinal cord injury, amyotrophic lateral sclerosis, Huntington""s chorea, Alzheimer""s and Parkinson""s diseases, cerebral palsy. Epilepsia may be, among other possibilities, treated by a local production, in the central nervous system, of the neurotransmitter GABA, as a result of the expression of the glutamic acid decarboxylase.
The invention is also of particular interest for the preparation of compositions for use in the treatment of hereditary diseases affecting the defective or deficient product of the mutant gene: lysosomal enzymes in lysosomal disses (e.g. hexosaminidases in Tay Sachs and Sandhoff diseases, arylsulfatase in metachromatic leucodystrophy, glucocerebrosidase in Gaucher""s disease, b-glucursoronidase in mucopolysaccharidosis, HGPRT in Lesh Nyhan disease, etc . . . ). Hereditary progressive neuron degenerations could be treated by transfer of the normal disease gene via adenoviral vectors, or, as discussed above, by induction of a local production of growth factors. For instance, it has been shown, that production of CNTF could slow down progressive motoneuronal degeneration (pmm) of mice (Sendtner et al, Nature 1992; 358: 502-504), the same being observed with aFGF on photoreceptor degeneration in inherited retinal dystrophy in rat (Faktorovich et al, Nature 1990; 347:83-86). Acquired spinal cord diseases like the frequent and constantly lethal Amyotrophic Lateral Sclerosis (ALS) could perhaps benefit from similar local production of CNTF that has been proved to protect motoneurons.
Inherited dysmyelinating diseases could also be improved by adenovirus-mediated gene transfer into myelin-synthesizing cells.
Finally, some other types of potential therapeutic agents could be locally produced into the CNS, for instance enkephalins to attenuate rebel pains, for instance in cancerous patients.
The invention also relates to the pharmaceutical compositions consisting of the recombinant adenoviral vectors containing the nucleotide sequences as defined above, in association with a pharmaceutical carrier suitable for the administration route to be selected, e.g. direct in situ injection of the viral suspensions obtained in the relevant neural tissue (or though far less preferred, through a general route, e.g. intravenous route, particularly when the adenoviral vector also contains a promoter selectively operative in determined nerve tissue or cells).
The invention is not limited to the therapeutical uses contemplated hereabove, of the adenoviral vectors. The latter can also, owing to their high infectivity, be used either in in vitro assays on determined populations of neural cells, e.g. for the sake of studying the capacity of a promoter (then coupled to a suitable xe2x80x9cmarkerxe2x80x9d, e.g. b-gal) of being recognized by the polymerases of said neural cells. Alternatively such adenoviral vectors can also be used for the detection or the evaluation of the interaction recombinant virus (that expressed by the nucleotide sequence under the control of a promoter operative in such cells) with a given population of neural cells or a more complex nerve tissue. For instance that evaluation or detection may aim at localizing those cells of the more complex tissue which carry a receptor for the virus. The detection can make use of any appropriate classical labeled method, e.g. use of labeled antibodies to detect expression products of the nucleotide sequence assayed. The prospects of these evaluations may be considerable in the field of neuroanatomy.
The recombinant vector of this invention is also useful in a method comprising causing the transcription product or expression product of a nucleotide sequence coding for a selected polypeptide to be targeted or produced in cells of the central nervous system, e.g. brain and spinal cord cells, particularly neural, glial or ependymal cells, of an animal and detecting the resulting physiological or behavioral modification induced in said animal by said transcription product or said expression product.
For instance in the case of a lesion of the septohippocampic track which depletes the hippocampus in acetylcholine, it would be possible to study the effects of the transfer of a gene coding for the choline acetyl transferase (CHAT) in the hippocampus. The introduction of that gene in the target core of cholinergic fibers (cut by the lesion) could elicit a re-increase of the amount of the available acetyl choline and, consequently, correct the deficit. This deficit can be evaluated by behavioral tests of memorization. For instance mice thrawn in a xe2x80x9cswimming poolxe2x80x9d can learn to find again a platform which enables them to escape to water (Morris swimming pool). However the animals whose septo-hippocampic track has been deteriorated are very handicapped in that operation. The detection of an increase of the acetyl choline produced as a result of the gene expression would then be appreciated by the ease with which the animals would be able again to find the platform.
According to another example, the invention would enable the analysis of the degree of compensation of troubles of the motor behavior produced by the denervation of the striatum, particularly in the event of a lesion of the dopaminergic cores of the mesencephale (substance nigra). The introduction of the gene coding for tyrosine hydroxylase (TH) in the striatum could correct that deficit The compensation can be evaluated by the study of the behavior of rats in the rotation test: animals wounded on one side only rotate in a repetitive fashion (over ten rotations per minute) when they receive an injection of apomorphine. The production of dopamine (linked to the introduction of the TH gene) could be appreciated in that behavioral test.
Still according to another example, the invention allows for the electrophysiological and behavioral study in other cases. For instance, the neurones of the dorsal horn of the spinal cord which transmit information bound to nociceptive stimulation (pain channels) are sensitive to morphine which causes their activity to be decreased. The introduction of a gene coding for an endomorphine in the spinal cord could provoke a secretion of this substance which, like morphine, would act of these cells. Such an action could be evaluated by behavioral tests (threshold of reaction of animals to nociceptive stimulation) as well as by electrophysiological studies on the neurones themselves of the spinal cord. Recording of the activity of these cells would enable one to appreciate the existence of a modulation induced by the expression of the transferred gene.
The possibilities afforded by the invention will be further illustrated, yet in a non-limitative manner by the description of a number of assays, which are in part supported by figures appended to the present description. Particularly, the Ad.RSVbgal appears as an appealing means to analyze neuronal and glialmorphology in specific areas of the brain by providing Golgi-like staining of cells at the injection site. Filing up of the axons by the enzyme may additionally provide ways to analyze projections of discrete neuronal populations. Conversely uptake of the virus by the terminals and subsequent retrograde transport to the neuronal cell body allows tracing of sets of afferents to a specific brain area.
One major advantage of this technique for neuroanatomy is the easy combination of the X-gal stain with all sorts of other labeling techniques, in particular immunocytochemistry. The use of vectors in which the nls sequence would be omitted might improve the efficacy of such a technique.
Similarly that adenoviral vector provides a basis for the study of the action of other promoters substituted for the RSV LTR promoter, with respect to different neural cell populations.